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EP4444751A1 - Immunothérapie pour le cancer - Google Patents

Immunothérapie pour le cancer

Info

Publication number
EP4444751A1
EP4444751A1 EP22843860.2A EP22843860A EP4444751A1 EP 4444751 A1 EP4444751 A1 EP 4444751A1 EP 22843860 A EP22843860 A EP 22843860A EP 4444751 A1 EP4444751 A1 EP 4444751A1
Authority
EP
European Patent Office
Prior art keywords
seq
nos
antibody
human
sema4a
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22843860.2A
Other languages
German (de)
English (en)
Inventor
Alexandra ADDYMAN
Georgina ANDERSON
Mark Austin
Michelle BARNARD
Denice Tsz Yau CHAN
Michael Chapman
Agata DIAMANDAKIS
Maria Groves
William Hawthorne
Stuart HAYNES
Lesley JENKINSON
Jean-Martin LAPOINTE
Kirsty-Jane MARTIN
Louise SLATER
Tristan Vaughan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MedImmune Ltd
Cancer Research Technology Ltd
Original Assignee
MedImmune Ltd
Cancer Research Technology Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MedImmune Ltd, Cancer Research Technology Ltd filed Critical MedImmune Ltd
Publication of EP4444751A1 publication Critical patent/EP4444751A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Cancer immunotherapy the induction of the immune system to attack tumour cells, has a long history, and has seen a recent resurgence of interest. This has been driven both by the success of immune checkpoint blockade and of cancer-directed immune therapies.
  • the latter exploiting the targeting of a cell surface protein on the cancer cell, is exemplified by monoclonal antibody therapies (e.g., rituximab, trastuzumab), introduced at the end of the twentieth century, and chimeric antigen receptor-T (CAR-T) cells, introduced at the start of the twenty-first.
  • monoclonal antibody therapies e.g., rituximab, trastuzumab
  • CAR-T chimeric antigen receptor-T
  • antigen escape i.e., the downregulation of the antigenic target by the cancer cell.
  • Antigen escape may be driven by intra-tumour heterogeneity, wherein subclones expressing low or absent levels of target protein gain a competitive growth advantage during immunotherapy.
  • novel immunotherapies should be targeted against antigens essential for cancer cell survival and combination treatments may be used.
  • Strategies which combine multiple monoclonal antibodies are particularly attractive, in part because of low toxicity, and in part because immunophenotyping of cancer cells can reveal susceptible and resistant sub-populations and lead to rational therapeutic decisions. There is thus an urgent need to identify novel target proteins on the surface of cancer cells.
  • WO2021195536 describes analysis of the expression of cell surface candidate targets in multiple myeloma (MM).
  • MM myeloma
  • Surface proteins of 7 different MM cell lines were biotinylated and subjected to spectrometry analysis thereby identifying 4761 proteins, an integrated database was used to generate cell surface molecule annotation, which was combined with exclusion based on expression levels to identify 326 surface proteins for further analysis by STRING.
  • a heatmap revealed protein annotation of 94 selected targets in several normal tissues and organs of the whole body. Molecules with high expression in any normal tissue except haematopoietic tissues and molecules with annotation in less than 2 out of 3 proteomic databases were excluded.
  • Anderson et al. (2022) describe plasma membrane profiling of primary human myeloma cells to identify cell surface proteins of a primary cancer.
  • SEMA4A semaphorin-4A
  • ADC antibody-drug conjugate
  • the antibody comprises a VH and a VL selected from the VH and VL of: a. 5Hg1-5Lg1 (SEQ ID NOS.: 20 and 29); b. 5Hg1-5Lg2 (SEQ ID NOS.: 38 and 47); c. 5Hg
  • 5Hg2-5Lg6 (SEQ ID NOS.: 128 and 137); h. 5Hg4-5Lg1 (SEQ ID NOS.: 146 and 155); i. 5Hg4-5Lg2 (SEQ ID NOS.: 164 and 173); j. 5Hg4-5Lg4 (SEQ ID NOS.: 182 and 191); k. 5Hg4-5Lg5 (SEQ ID NOS.: 200 and 209); l. 5Hg5-5Lg2 (SEQ ID NOS.: 218 and 227); m. C0120903 (SEQ ID NOS.: 236 and 245); n. C0120904 (SEQ ID NOS.: 254 and 263); o.
  • C0120905 (SEQ ID NOS.: 272 and 281); p. C0120906 (SEQ ID NOS.: 290 and 299); q. C0120910 (SEQ ID NOS.: 308 and 317); r. C0120913 (SEQ ID NOS.: 326 and 335); s. C0120914 (SEQ ID NOS.: 344 and 353); t. C0120917 (SEQ ID NOS.: 362 and 371); u. C0120918 (SEQ ID NOS.: 380 and 389); v. C0120919 (SEQ ID NOS.: 398 and 407); w.
  • C0120920 (SEQ ID NOS.: 416 and 425); x. C0120921 (SEQ ID NOS.: 434 and 443); y. C0120922 (SEQ ID NOS.: 452 and 461); z. C0120923 (SEQ ID NOS.: 470 and 479); aa. C0120924 (SEQ ID NOS.: 488 and 497); bb. C0120925 (SEQ ID NOS.: 506 and 515); cc. C0120926 (SEQ ID NOS.: 524 and 533); dd. C0120927 (SEQ ID NOS.: 542 and 551); ee.
  • C0120928 (SEQ ID NOS.: 560 and 569); ff. C0120929 (SEQ ID NOS.: 578 and 587); and gg. C0120930 (SEQ ID NOS.: 596 and 605); wherein the sequences are defined by Kabat nomenclature. 4.
  • cytotoxin is selected from monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE, vedotin) and mertansine (DM1, e.g., as emtansine with an SMCC linker).
  • MMAF monomethyl auristatin F
  • MMAE monomethyl auristatin E
  • DM1 mertansine
  • linker is a non- cleavable linker or is a cleavable linker.
  • cytotoxin is monomethyl auristatin E (MMAE) and the linker is a mc-vcPAB linker (malemide-based linker, cysteine linked) or the cytotoxin is mertansine (DM1) and the linker is a SMCC linker (NHS-ester based, lysine).
  • MMAE monomethyl auristatin E
  • DM1 mertansine
  • SMCC linker NHS-ester based, lysine
  • a human or humanised anti-human SEMA4A antibody comprising a VH comprising HCDR1, HCDR2 and HCDR3 and a VL comprising LCDR1, LCDR2 and LCDR3, wherein the CDRs are selected from the CDRs of: (a) 5E3 (SEQ ID NOS.: 3, 4, 5, 12, 13, 14); (b) 5Hg1-5Lg1 (SEQ ID NOS.: 21, 22, 23, 30, 31, 32); (c) 5Hg1-5Lg2 (SEQ ID NOS.: 39, 40, 41, 48, 49, 50); (d) 5Hg2-5Lg1 (SEQ ID NOS.: 57, 58, 59, 66, 67, 68); (e) 5Hg2-5Lg2 (SEQ
  • an antibody according to clause 10 wherein the antibody comprises a VH and a VL selected from the VH and VL of: (a) 5Hg1-5Lg1 (SEQ ID NOS.: 20 and 29); (b) 5Hg1-5Lg2 (SEQ ID NOS.: 38 and 47); (c) 5Hg2-5Lg1 (SEQ ID NOS.: 56 and 65); (d) 5Hg2-5Lg2 (SEQ ID NOS.: 74 and 83); (e) 5Hg2-5Lg3 (SEQ ID NOS.: 92 and 101); (f) 5Hg2-5Lg5 (SEQ ID NOS.: 110 and 119); (g) 5Hg2-5Lg6 (SEQ ID NOS.: 128 and 137); (h) 5Hg4-5Lg1 (SEQ ID NOS.: 146 and 155); (i) 5Hg4-5Lg2 (SEQ ID NOS.: 164 and 173); (j) 5Hg4-5Lg
  • a chimeric antigen receptor comprising an antigen-binding domain of the monoclonal antibody of any one of clauses 10 or 11 linked to a T-cell activation moiety.
  • a composition comprising an ADC according to any one of clauses 1 to 9, or an anti- human SEMA4A antibody according to any one of clauses 10 or 11, or a CAR of any one of clauses 12 and 13 and a diluent.
  • MM multiple myeloma
  • NHL non-Hodgkin’s lymphoma
  • AML acute myeloid leukaemia
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • a medicament for the therapeutic treatment of a cancer for use in the manufacture of a medicament for the therapeutic treatment of a cancer; f. for use in the manufacture of a medicament for the therapeutic treatment of a haematological cancer; g. for use in the manufacture of a medicament for the therapeutic treatment of a haematological cancer selected from MM, NHL, AML, DLBCL and FL, or, h. for inducing cell death in cells expressing SEMA4A at the cell surface. 16.
  • a method of treatment of a cancer such as a haematological cancer, e.g., a haematological cancer selected from MM, NHL, AML, DLBCL and FL, comprising administration of an ADC according to any one of clauses 1 to 9, an antibody according to any one of clauses 10 or 11, a CAR according to any one of clauses 12 or 13 or a composition according to clause 14, to a subject.
  • a method for manufacture of an ADC according to any one of clauses 1 to 9 comprising conjugation of an antibody according to clause 10 or clause 11 to a cytotoxin via a linker. 18.
  • An isolated recombinant DNA or RNA sequence comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof, according to any one of clauses 10 or 11. 19.
  • An isolated recombinant DNA sequence of clause 18 which is a vector.
  • 20. An isolated recombinant DNA sequence of clause 18 or clause 19 which is an expression vector.
  • 21. An isolated recombinant DNA sequence of any one of clauses 18 to 20 encoding an antibody or antigen-binding fragment thereof, according to any one of clauses 10 or 11 under control of a promoter.
  • 22. A host cell comprising a DNA or RNA sequence according to any one of clauses 18 to 21. 23.
  • a host cell of clause 22 capable of expressing an isolated antibody or antigen-binding fragment thereof, of any one of clauses 10 or 11. 24.
  • a method of making an isolated antibody or antigen-binding fragment thereof, of clause 10 or 11 comprising culturing a host cell according to clause 22 or 23 in conditions suitable for expression of the isolated antibody or antigen-binding fragment thereof.
  • the present invention provides an antibody-drug conjugate (ADC) comprising a human or humanised antibody, or an antigen-binding fragment thereof, directed against human Semaphorin4A (SEMA4A) conjugated to a cytotoxin.
  • ADC antibody-drug conjugate
  • SEMA4A human Semaphorin4A
  • antibody-drug conjugate refers to a compound comprising an antibody, such as a humanised or human monoclonal antibody (mAb) or an antigen-binding fragment thereof attached to a cytotoxic agent (generally a small molecule drug with a high systemic toxicity) via a chemical linker.
  • a cytotoxic agent generally a small molecule drug with a high systemic toxicity
  • an ADC may comprise a small molecule cytotoxin that has been chemically modified to contain a linker. The linker is then used to conjugate the cytotoxin to the antibody, or antigen-binding fragment thereof.
  • the cytotoxin upon binding to the target antigen on the surface of the cell, the cytotoxin can be released by cleavage of the linker or proteolysis, the cytotoxin can then bind to its target and induce cell death.
  • the ADCs described herein may comprise a whole antibody or an antibody fragment.
  • a whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide.
  • Each of the heavy chains contains one N-terminal variable (V H ) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable (V L ) region and one C- terminal constant (CL) region.
  • variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • the V H and V L regions have the same general structure, with each region comprising four framework regions, whose sequences are relatively conserved.
  • the framework regions are connected by three complementarity determining regions (CDRs).
  • the three CDRs known as CDR1, CDR2, and CDR3, form the "hypervariable region" of each variable domain, which is responsible for antigen binding.
  • the ADC may comprise an antigen-binding fragment of a humanised or human antibody.
  • antibody fragment refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen.
  • the antibody fragment may comprise, for example, one or more CDRs, the variable region (or portions thereof), or combinations thereof, optionally in further combination with the constant region (or portions thereof).
  • antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and
  • Antibody fragments are known in the art and are described in more detail in, e.g., U.S. Patent Application Publication 2009/0093024 A1.
  • An ADC of the invention comprising a humanised antibody of the invention or comprising a human antibody of the invention is capable of binding specifically to recombinant human SEMA4A (UniProt ID Q9H3S1), and may also bind to recombinant cynomolgus SEMA4A (UniProt ID G7NV79) and/or to recombinant mouse SEMA4A (UniProt ID Q62178).
  • the ADC comprises a variable region of a humanised or fully human anti- human-SEMA4A antibody.
  • the ADC may comprise a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of an anti-human-SEMA4A monoclonal antibody.
  • the ADC comprises a light chain variable region and a heavy chain variable region of an anti-human-SEMA4A antibody.
  • cytotoxin and cytotoxic agent refer to any molecule that inhibits or prevents the function of cells and/or causes destruction of cells (cell death), and/or exerts anti-proliferative effects. It will be appreciated that a cytotoxin or cytotoxic agent of an ADC also is referred to in the art as the "payload” or “warhead” of the ADC.
  • cytotoxic agents A number of classes of cytotoxic agents are known in the art to have potential utility in ADC molecules and can be used in the ADC described herein.
  • An exemplary class of cytotoxic agents includes anti-microtubule agents such as a tubulysin, a maytansinoid, an auristatin, or derivatives thereof. More specifically, the cytotoxic agent may be, for example MMAF, MMAE or DM1, DM4. In one embodiment, the cytotoxic agent may be an anti-microtubule agent.
  • anti- microtubule agent and "microtubule-targeting agent,” are synonymous and refer to an agent that inhibits cell division by interfering with microtubules.
  • Tubulysins are members of a class of natural products isolated from myxobacterial species (Sasse et al., 2000) which act as mitotic poisons that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis (Steinmetz et al., 2004; Khalil et al., 2006; Kaur et al., 2006).
  • Examples of tubulysins are disclosed in, for example, International Patent Application Publication Nos. WO 2015/157594, WO 2004/005326, WO 2012/019123, WO 2009/134279, WO 2009/055562, WO 2004/005327; U.S.
  • Patents 7,776,841, 7,754,885, and 7,816,377 and U.S. Patent Application Publications 2010/0240701, 2011/0021568, and 2011/0263650.
  • Maytansinoids inhibit polymerization of the microtubule protein tubulin, thereby preventing formation of microtubules (see, e.g., U.S. Patent No.6,441,163 and Remillard et al., 1975). Maytansinoids have been shown to inhibit tumour cell growth in vitro using cell culture models, and in vivo using laboratory animal systems.
  • cytotoxicity of maytansinoids is 1,000-fold greater than conventional chemotherapeutic agents, such as, for example, methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Patent 5,208,020).
  • Maytansinoids include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogues and derivatives (see, e.g., U.S. Patents 5,208,020 and 6,441,163).
  • C-3 esters of maytansinol can be naturally occurring or synthetically derived.
  • C-3 maytansinol esters can be classified as a C-3 ester with simple carboxylic acids, or a C-3 ester with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former.
  • Synthetic maytansinoid analogues also are known in the art and described in, for example, Kupchan et al., 1978. Methods for generating maytansinol and analogues and derivatives thereof are described in, for example, U.S. Patent 4,151,042.
  • Examples of maytansinoids that may be used in connection with the ADC described herein include, but are not limited to, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) and N2'- deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).
  • Auristatins represent a class of highly potent anti-mitotic agents that have shown substantial preclinical activity at well-tolerated doses (Law et al., 2006; Ma et al., 2006; Tse et al., 2006; Oflazoglu et al., 2008, Oflazoglu et al., 2008). Auristatin ADCs are currently being evaluated in preclinical and clinical trials.
  • auristatins examples include, but are not limited to, monomethyl auristatin E (MMAE) and the related molecule monomethyl auristatin F (MMAF) (see, e.g., Doronina et al., 2003; Doronina et al., 2006).
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • SEMA4A humanised or human monoclonal antibody, or antigen-binding fragment thereof may be conjugated to a cytotoxin using any suitable method known in the art, including site- specific or non-site specific conjugation methods.
  • the antibody or antigen-binding fragment thereof is randomly conjugated to a cytotoxic agent, for example, by partial reduction of the antibody or antibody fragment, followed by reaction with a desired agent with or without a linker moiety attached.
  • a cytotoxic agent for example, by partial reduction of the antibody or antibody fragment, followed by reaction with a desired agent with or without a linker moiety attached.
  • the antibody or antigen-binding fragment thereof may be reduced using dithiothreitol (DTT) or a similar reducing agent.
  • DTT dithiothreitol
  • the cytotoxic agent with or without a linker moiety attached thereto, can then be added at a molar excess to the reduced antibody or antibody fragment in the presence of dimethyl sulfoxide (DMSO). After conjugation, excess free cysteine may be added to quench unreacted agent. The reaction mixture may then be purified and buffer-exchanged into phosphate buffered saline (PBS).
  • the cytotoxic agent may be conjugated to the SEMA4A monoclonal antibody using site-specific conjugation methods at specific reactive amino acid residues, yielding homogeneous ADC preparations with uniform stoichiometry. Site-specific conjugation may be through a cysteine residue or a non-natural amino acid.
  • the cytotoxic agent may be conjugated to the antibody, or antigen binding fragment thereof, through at least one cysteine residue.
  • a cytotoxic agent may be chemically conjugated to the side chain of an amino acid at a specific Kabat position (Kabat et al., 1991) in the Fc region of the SEMA4A monoclonal antibody.
  • the cytotoxic agent may be conjugated to the SEMA4A monoclonal antibody through a cysteine residue at any suitable position in the Fc region of the antibody.
  • the cytotoxic agent may be conjugated to the SEMA4A monoclonal antibody or antigen binding fragment thereof through a thiol-maleimide linkage, such as, for example, via a sulfhydryl reactive group at the hinge and heavy-light chains.
  • the SEMA4A humanised or human monoclonal ADC described herein comprises at least one cytotoxin molecule conjugated thereto; however, the SEMA4A humanised or human monoclonal antibody may comprise any suitable number of cytotoxin molecules conjugated thereto (e.g., 1, 2, 3, 4, or more cytotoxin molecules) to achieve a desired therapeutic effect.
  • an ADC of the invention may have a drug-antibody ratio (DAR) of, for example, 1, 2, 3, 4, 5, 6, 7, or 8.
  • DAR is the average drug (cytotoxin) to antibody ratio for a given preparation of ADC.
  • DAR is a measure of drug loading for an ADC.
  • the invention provides a humanised or human monoclonal antibody, or an antigen-binding fragment thereof, directed against human SEMA4A described above independent of an ADC.
  • Humanised and human antibodies of the invention and ADC or CAR comprising such antibodies are capable of binding specifically to recombinant human SEMA4A (UniProt ID Q9H3S1, SEQ ID NO: 613) and may also bind recombinant cynomolgus SEMA4A (UniProt ID G7NV79, SEQ ID NO: 614) and/or recombinant mouse SEMA4A (UniProt ID Q62178, SEQ ID NO: 615).
  • the humanised or human antibody, or an antigen-binding fragment thereof, directed against human SEMA4A may comprise any suitable binding affinity to human SEMA4A or an epitope thereof.
  • affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (K D ).
  • K D dissociation constant
  • the affinity of an antibody or antigen-binding fragment thereof for an antigen or epitope of interest can be measured using any method known in the art. Such methods include, for example, fluorescence activated cell sorting (FACS), surface plasmon resonance (e.g., Biacore, ProteOn), biolayer interferometry (BLI, e.g., Octet), kinetics exclusion assay (e.g., KinExA), separable beads (e.g., magnetic beads), antigen panning, and/or enzyme-linked immunosorbent assay (ELISA) (Janeway et al., 2001).
  • FACS fluorescence activated cell sorting
  • surface plasmon resonance e.g., Biacore, ProteOn
  • biolayer interferometry e.g., Octet
  • KinExA kinetic
  • binding affinity of a particular antibody will vary depending on the method that is used to analyze the binding affinity.
  • Affinity of a binding agent to a ligand, such as affinity of an antibody for an epitope can be, for example, from about 1 nM to about 100 nM.
  • the monoclonal antibody or an antigen-binding fragment thereof may bind to human SEMA4A with a K D less than or equal to 500, 400, 300, 200 or 100 nanomolar (e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values).
  • a K D less than or equal to 500, 400, 300, 200 or 100 nanomolar (e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values).
  • the monoclonal antibody may bind to human SEMA4A with a K D less than or equal to 10 nanomolar (e.g., about 9 nM, about 8.5 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, or a range defined by any two of the foregoing values).
  • 10 nanomolar e.g., about 9 nM, about 8.5 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM,
  • An antigen-binding portion or fragment of a humanised or human antibody of the invention can be of any size so long as the portion binds to human SEMA4A.
  • An antibody or antigen-binding fragment thereof of the invention may be produced by recombinant means.
  • a “recombinant antibody” is an antibody which has been produced by a recombinantly engineered host cell.
  • An antibody or antigen-binding fragment thereof in accordance with the invention is optionally isolated or purified.
  • the term “antibody” or “antibody molecule” describes an immunoglobulin whether natural or partly or wholly synthetically produced.
  • An antigen-binding protein of the invention may be an antibody, preferably a monoclonal antibody, and may be human or non-human, chimeric or humanised.
  • the antibody molecule is preferably a monoclonal antibody molecule.
  • antibodies are the immunoglobulin isotypes, such as immunoglobulin G, and their isotypic subclasses, such as IgG1, IgG2, IgG3 and IgG4, as well as fragments thereof.
  • the four human subclasses (IgG1, IgG2, IgG3 and IgG4) each contain a different heavy chain; but they are highly homologous and differ mainly in the hinge region and the extent to which they activate the host immune system.
  • IgG1 and IgG4 contain two inter-chain disulphide bonds in the hinge region, IgG2 has 4 and IgG3 has 11 inter-chain disulphide bonds.
  • antibody and antibody molecule include antibody fragments, such as Fab and scFv fragments, provided that said fragments comprise a CDR-based antigen binding site for an epitope of human SEMA4A.
  • antibody fragments include but are not limited to Fv, Fab, F(ab'), Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv) and domain antibodies (sdAbs).
  • antigen-binding protein “antibody” or “antibody molecule”, as used herein, is thus equivalent to “antibody or antigen-binding fragment thereof”.
  • Antibodies are immunoglobulins, which have the same basic structure consisting of two heavy and two light chains forming two Fab arms containing identical domains that are attached by a flexible hinge region to the stem of the antibody, the Fc domain, giving the classical ‘Y’ shape.
  • the Fab domains consist of two variable and two constant domains, with a variable heavy (VH) and constant heavy 1 (CH1) domain on the heavy chain and a variable light (VL) and constant light (CL) domain on the light chain.
  • the two variable domains (VH and VL) form the variable fragment (Fv), which provides the CDR-based antigen specificity of the antibody, with the constant domains (CH1 and VL) acting as a structural framework.
  • Each variable domain contains three hypervariable loops, known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the three CDRs (CDR1, CDR2, and CDR3) are flanked by four less-variable framework (FW) regions (FW1, FW2, FW3 and FW4) to give a structure FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.
  • FW less-variable framework
  • the CDRs provide a specific antigen recognition site on the surface of the antibody. Both Kabat and ImMunoGeneTics (IMGT) numbering nomenclature may be used herein.
  • amino acid residues are numbered herein according to the Kabat numbering scheme (Kabat et al., 1991). For those instances when the IMGT numbering scheme is used, amino acid residues are numbered herein according to the ImMunoGeneTics (IMGT) numbering scheme described in Lefranc et al., 2005.
  • IMGT ImMunoGeneTics
  • CDRs of one immunoglobulin into another immunoglobulin are described for example in EP-A-184187, GB2188638A or EP-A-239400.
  • a hybridoma or other cell producing an antibody molecule may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
  • Antibody humanisation involves the transfer, or “grafting”, of critical non-human amino acids onto a human antibody framework. Primarily this includes the grafting of amino acids in the complementarity-determining regions (CDRs), but potentially also other framework amino acids critical for the V H – V L interface and for orientation of the CDRs.
  • CDRs complementarity-determining regions
  • Humanisation seeks to introduce human content to reduce the risk of immunogenicity, while retaining the original binding activity of the non-human parental antibody.
  • the term "humanised antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences; optionally additional framework region modifications can be made within the human framework sequences.
  • humanised antibody includes antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences and optimised (for example by affinity maturation), e.g., by modification of one or more amino acid residues in one or more of the CDRs and/or in one or more framework sequence to modulate or improve a biological property of the humanised antibody, e.g., to increase affinity, or to modulate the on rate and/or off rate for binding of the antibody to its target epitope.
  • Variable domains employed in the invention may be obtained or derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus or actual sequences of known human variable domains.
  • a repertoire of variable domains may be displayed in a suitable host system, such as the phage display system of WO92/01047, which is herein incorporated by reference in its entirety, or any of a subsequent large body of literature, including Kay, Winter & McCafferty [Kay, B.K., Winter, J., and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press], so that suitable binding members may be selected.
  • Other suitable host systems include, but are not limited to, yeast display, bacterial display, T7 display, viral display, cell display, ribosome display and covalent display.
  • the term “antigen-binding protein” or “antibody” should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, an aptamer, affimer or bicyclic peptide, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A- 0120694 and EP-A-0125023.
  • an antibody fragment comprising both CDR sequences and CH3 domain is a minibody, which comprises a scFv joined to a CH3 domain (Hu et al., 1996).
  • a domain (single-domain) antibody is a peptide, usually about 110 amino acids long, comprising one variable domain (V H ) of a heavy-chain antibody, or of an IgG.
  • a single-domain antibody (sdAb), (e.g., nanobody), is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody (comprising two heavy and two light chains), it is an antigen-binding protein able to bind selectively to a specific antigen.
  • Domain antibodies have a molecular weight of only 12–15 kDa and are thus much smaller than antibodies composed of two heavy protein chains and two light chains (150–160 kDa), and domain antibodies are even smaller than Fab fragments ( ⁇ 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments ( ⁇ 25 kDa, two variable domains, one from a light and one from a heavy chain).
  • Single-domain antibodies have been engineered from heavy- chain antibodies found in camelids; these are termed V HH fragments.
  • Cartilaginous fish also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained.
  • a domain (single-domain) antibody may be a V H or V L .
  • a domain antibody may be a V H or V L of human or murine origin. Although most single-domain antibodies are heavy chain variable domains, light chain single- domain antibodies (V L ) have also been shown to bind specifically to target epitopes.
  • Protein scaffolds have relatively defined three-dimensional structures and typically contain one or more regions which are amenable to specific or random amino acid sequence variation, to produce antigen-binding regions within the scaffold that are capable of binding to an antigen.
  • a humanised or human antibody or antigen-binding fragment of the invention binds specifically to human SEMA4A.
  • the term “specific” may refer to the situation in which the antibody molecule will not show any significant binding to molecules other than its specific binding partner(s), here an epitope of human SEMA4A.
  • the term “specific” is also applicable where the antibody is specific for particular epitopes, such as an epitope of human SEMA4A that is carried by a number of antigens in which case the antibody molecule will be able to bind to the various antigens carrying the epitope.
  • the epitope may be present in human SEMA4A expressed on the cell surface or soluble SEMA4A (sSEMA4A) shed from the cell surface or expressed recombinantly.
  • a humanised or human antibody or antigen- binding fragment of the invention binds specifically to human SEMA4A and binds to cynomolgus and/or mouse SEMA4A, accordingly, a humanised or human antibody or antigen- binding fragment of the invention may bind specifically to human SEMA4A and be cross reactive with cynomolgus and/or mouse SEMA4A.
  • the humanised antibodies and antigen-binding fragments thereof are humanised versions of mouse 5E3 mAb (Cat. #148402, BioLegend, Inc., USA), comprising the set of six CDRs of mouse 5E3 (SEQ ID NOS.: 3, 4, 5, 12, 13 and 14) and human framework sequences.
  • humanised and human antibodies and antigen-binding fragments thereof of the invention bind to an epitope bound by mouse 5E3 mAb (Cat. #148402, BioLegend, Inc., USA) or compete with the mouse mAb 5E3 for binding to an epitope on SEMA4A, preferably human SEMA4A.
  • Amino acids may be referred to by their one letter or three letter codes, or by their full name. The one and three letter codes, as well as the full names, of each of the twenty standard amino acids are set out below in Table 1.
  • Amino acid One letter code Three letter code Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamic acid E Glu Glutamine Q Gln Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val Table 1. Amino acids, one and three-letter codes.
  • the invention provides a humanised or human antibody or an antigen-binding fragment thereof comprising the set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a clone selected from: a) 5E3 (SEQ ID NOS.: 3, 4, 5, 12, 13, 14); b) 5Hg1-5Lg1 (SEQ ID NOS.: 21, 22, 23, 30, 31, 32); c) 5Hg1-5Lg2 (SEQ ID NOS.: 39, 40, 41, 48, 49, 50); d) 5Hg2-5Lg1 (SEQ ID NOS.: 57, 58, 59, 66, 67, 68); e) 5Hg2-5Lg2 (SEQ ID NOS.: 75, 76, 77, 84, 85, 86); f) 5Hg2-5Lg3 (SEQ ID NOS.: 93, 94, 95, 102, 103, 104); g) 5E3 (S
  • the CDRs are spaced by framework regions FW1, FW2, FW3 and FW4, to give a structure in the format FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.
  • the invention provides a human or humanised antibody or an antigen-binding fragment thereof comprising a VH and/or VL sequence of a clone selected from: a) 5Hg1-5Lg1 (SEQ ID NOS.: 20 and 29); b) 5Hg1-5Lg2 (SEQ ID NOS.: 38 and 47); c) 5Hg2-5Lg1 (SEQ ID NOS.: 56 and 65); d) 5Hg2-5Lg2 (SEQ ID NOS.: 74 and 83); e) 5Hg2-5Lg3 (SEQ ID NOS.: 92 and 101); f) 5Hg2-5Lg5 (SEQ ID NOS.: 110 and 119); g) 5Hg2-5Lg6 (SEQ ID NO
  • antibodies were selected for germlining.
  • the amino acid sequences of the V H and V L domains of the antibodies were compared to human germline V, D and J regions accessible via IMGT (ImMunoGeneTics; www.imgt.org) or ImmuneDiscover databases and the closest germline was identified by sequence similarity.
  • the germlining process consisted of reverting framework residues in the V H and V L domains to the closest germline sequence to identically match human antibodies.
  • An antibody or an antigen-binding fragment thereof of the invention may comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 further amino acid modifications in the V H and/or V L sequences, provided that functional properties of the antibody are retained.
  • a modification may be an amino acid substitution, deletion or insertion; preferably, the modification is a substitution.
  • the substitutions may be conservative substitutions, for example according to Table 2.
  • amino acids in the same category in the middle column are substituted for one another, i.e., a non-polar amino acid is substituted with another non-polar amino acid, for example.
  • amino acids in the same line in the rightmost column are substituted for one another.
  • substitution(s) may be functionally conservative.
  • an antibody or an antigen-binding fragment thereof of the invention may comprise a V H and/or V L domain sequence with one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), preferably 20 alterations or fewer, 15 alterations or fewer, 10 alterations or fewer, 5 alterations or fewer, 4 alterations or fewer, 3 alterations or fewer, 2 alterations or fewer, or 1 alteration compared with the V H and/or V L sequences of the invention set forth herein.
  • the invention provides a humanised or human antibody or an antigen-binding fragment thereof comprising a V H and/or V L domain with an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VH and/or VL amino acid sequence of a clone selected from: a) 5E3 (SEQ ID NOS.: 2 and 11); b) 5Hg1-5Lg1 (SEQ ID NOS.: 20 and 29); c) 5Hg1-5Lg2 (SEQ ID NOS.: 38 and 47); d) 5Hg2-5Lg1 (SEQ ID NOS.: 2
  • a humanised or human antibody or an antigen- binding fragment thereof of the invention comprises a VH domain amino acid sequence comprising the set of HCDRs: HCDR1, HCDR2, and HCDR3, and/or a VL domain amino acid sequence comprising the set of LCDRs: LCDR1, LCDR2, and LCDR3 of a clone selected from: (a) 5E3 (SEQ ID NOS.: 3, 4, 5, 12, 13, 14); (b) 5Hg1-5Lg1 (SEQ ID NOS.: 21, 22, 23, 30, 31, 32); (c) 5Hg1-5Lg2 (SEQ ID NOS.: 39, 40, 41, 48, 49, 50); (d) 5Hg2-5Lg1 (SEQ ID NOS.: 57, 58, 59, 66, 67, 68); (e) 5Hg2-5Lg2 (SEQ ID NOS.: 75, 76, 77, 84, 85, 86); (f) 5Hg
  • 5E3 (SEQ ID NOS.: 2 and 11); b. 5Hg1-5Lg1 (SEQ ID NOS.: 20 and 29); c. 5Hg1-5Lg2 (SEQ ID NOS.: 38 and 47); d. 5Hg2-5Lg1 (SEQ ID NOS.: 56 and 65); e. 5Hg2-5Lg2 (SEQ ID NOS.: 74 and 83); f. 5Hg2-5Lg3 (SEQ ID NOS.: 92 and 101); g. 5Hg2-5Lg5 (SEQ ID NOS.: 110 and 119); h. 5Hg2-5Lg6 (SEQ ID NOS.: 128 and 137); i.
  • 5Hg4-5Lg1 (SEQ ID NOS.: 146 and 155); j. 5Hg4-5Lg2 (SEQ ID NOS.: 164 and 173); k. 5Hg4-5Lg4 (SEQ ID NOS.: 182 and 191); l. 5Hg4-5Lg5 (SEQ ID NOS.: 200 and 209); m. 5Hg5-5Lg2 (SEQ ID NOS.: 218 and 227); n. C0120903 (SEQ ID NOS.: 236 and 245); o. C0120904 (SEQ ID NOS.: 254 and 263); p. C0120905 (SEQ ID NOS.: 272 and 281); q.
  • C0120906 (SEQ ID NOS.: 290 and 299); r. C0120910 (SEQ ID NOS.: 308 and 317); s. C0120913 (SEQ ID NOS.: 326 and 335); t. C0120914 (SEQ ID NOS.: 344 and 353); u. C0120917 (SEQ ID NOS.: 362 and 371); v. C0120918 (SEQ ID NOS.: 380 and 389); w. C0120919 (SEQ ID NOS.: 398 and 407); x. C0120920 (SEQ ID NOS.: 416 and 425); y.
  • C0120921 (SEQ ID NOS.: 434 and 443); z. C0120922 (SEQ ID NOS.: 452 and 461); aa. C0120923 (SEQ ID NOS.: 470 and 479); bb. C0120924 (SEQ ID NOS.: 488 and 497); cc. C0120925 (SEQ ID NOS.: 506 and 515); dd. C0120926 (SEQ ID NOS.: 524 and 533); ee. C0120927 (SEQ ID NOS.: 542 and 551); ff. C0120928 (SEQ ID NOS.: 560 and 569); gg.
  • C0120929 (SEQ ID NOS.: 578 and 587); and hh. C0120930 (SEQ ID NOS.: 596 and 605); when defined by Kabat nomenclature. Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty equalling 12 and a gap extension penalty equalling 4.
  • GAP GAP polypeptide sequence alignment
  • other algorithms e.g., BLAST (which uses the method of Altschul et al., 1990), FASTA (which uses the method of Pearson and Lipman 1988), or the Smith-Waterman algorithm (Smith and Waterman 1981), or the TBLASTN program, of Altschul et al., (1990) supra, generally employing default parameters.
  • the psi-Blast algorithm may be used (Altschul et al., 1997). Sequence alignments may also be performed using CLUSTAL (W) algorithm.
  • the antibody may comprise a CH2 domain.
  • the CH2 domain is preferably located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule.
  • the CH2 domain of the antibody is preferably the CH2 domain of human IgG1, IgG2, IgG3, or IgG4, more preferably the CH2 domain of human IgG1.
  • the sequences of human IgG domains are known in the art.
  • the antibody may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain.
  • the immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer.
  • the hinge region, or part thereof is a human IgG1, IgG2, IgG3 or IgG4 hinge region, or part thereof. More preferably, the hinge region, or part thereof, is an IgG1 hinge region, or part thereof.
  • the sequence of the CH3 domain is not particularly limited.
  • the CH3 domain is a human immunoglobulin G domain, such as a human IgG1, IgG2, IgG3, or IgG4 CH3 domain, most preferably a human IgG1 CH3 domain.
  • An antibody of the invention may comprise a human IgG1, IgG2, IgG3, or IgG4 constant region.
  • the sequences of human IgG1, IgG2, IgG3, or IgG4 CH3 domains are known in the art.
  • An antibody of the invention may comprise a human IgG constant region, e.g., a human IgG1 constant region.
  • An antibody of the invention may comprise a human IgG Fc that has been modified to permit conjugation with a linker and cytotoxin.
  • An antibody of the invention may comprise a human IgG heavy chain, such as a human IgG1 heavy chain, engineered to contain one or more, e.g., 2, site-specific engineered cysteines that enable conjugation of cytotoxin to the antibody via a linker in a controlled manner as described in Dimasi et al.,2017; Li et al., 2016 and Gallagher et al., 2019 (e.g., a human IgG1 heavy chain with 239iCys and S442C (EU numbering) in the CH2 and CH3 Fc domain, respectively to generate a drug to antibody ratio of 4 (human IgG1 Alya).
  • a human IgG heavy chain such as a human IgG1 heavy chain, engineered to contain one or more, e.g., 2, site-specific engineered cysteines
  • An antibody of the invention may comprise a human lambda light chain constant domains or human kappa light chain constant domains.
  • An antibody of the invention may comprise a human IgG Fc with effector function.
  • Fc receptors FcRs
  • FcRs are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including Fc ⁇ R (IgG), Fc ⁇ RI (IgE), Fc ⁇ RI (IgA), Fc ⁇ R (IgM) and Fc ⁇ R (IgD).
  • Fc ⁇ RI Fc ⁇ RI
  • CD32 Fc ⁇ RIIa, Fc ⁇ RIIb and Fc ⁇ RIIc
  • CD16 Fc ⁇ RIIIa and Fc ⁇ RIIIb
  • Fc ⁇ RI is classed as a high affinity receptor (nanomolar range affinity) while Fc ⁇ RII and Fc ⁇ RIII are low to intermediate affinity (micromolar range affinity).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fc ⁇ Rs on the surface of effector cells naturally killer cells, macrophages, monocytes and eosinophils
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCC effector function varies depending on the specific IgG subtype. Although the level of variation is dependent on the allotype and specific Fc ⁇ R, in simple terms ADCC effector function is high for human IgG1 and IgG3, and low for IgG2 and IgG4. See Table 3 below for IgG subtype variation in effector functions, ranked in decreasing potency. E A C Table 3. IgG subtype variation in effector functions, ranked in decreasing potency.
  • Fc ⁇ Rs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has resulted in engineering efforts to modulate IgG effector functions.
  • Antibodies of the invention may have an Fc with effector function, enhanced effector function or with reduced effector function.
  • the potency of antibodies can be increased by enhancement of the ability to mediate cellular cytotoxicity functions, such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP).
  • ADCC antibody-dependent cell-mediated phagocytosis
  • a number of mutations within the Fc domain have been identified that either directly or indirectly enhance binding of Fc receptors and significantly enhance cellular cytotoxicity: the mutations S239D/A330L/I332E (“3M”), F243L or G236A.
  • effector function can be achieved by modifying the glycosylation of the Fc domain, Fc ⁇ Rs interact with the carbohydrates on the CH2 domain and the glycan composition has a substantial effect on effector function activity.
  • Afucosylated (non-fucosylated) antibodies exhibit greatly enhanced ADCC activity through increased binding to Fc ⁇ RIIIa.
  • Activation of ADCC and CDC may be desirable for some therapeutic antibodies, however, in some embodiments, an antibody that does not activate effector functions is preferred.
  • IgG4 antibodies are the preferred IgG subclass for receptor blocking without cell depletion. However IgG4 molecules can exchange half- molecules in a dynamic process termed Fab-arm exchange.
  • This phenomenon can occur between therapeutic antibodies and endogenous IgG4.
  • the S228P mutation has been shown to prevent this recombination process allowing the design of IgG4 antibodies with a reduced propensity for Fab-arm exchange.
  • Fc engineering approaches have been used to determine the key interaction sites for the IgG1 Fc domain with Fc ⁇ receptors and C1q and then mutate these positions to reduce or abolish binding. Through alanine scanning the binding site of C1q to a region covering the hinge and upper CH2 of the Fc domain was identified.
  • the CH2 domain of an antibody or fragment of the invention may comprise one or more mutations to decrease or abrogate binding of the CH2 domain to one or more Fc ⁇ Rs, such as Fc ⁇ RI, Fc ⁇ Rlla, Fc ⁇ Rllb, Fc ⁇ RIII and/or to complement.
  • Fc ⁇ Rs such as Fc ⁇ RI, Fc ⁇ Rlla, Fc ⁇ Rllb, Fc ⁇ RIII and/or to complement.
  • CH2 domains of human lgG domains normally bind to Fc ⁇ Rs and complement, decreased binding to Fc ⁇ Rs is expected to decrease antibody-dependent cell-mediated cytotoxicity (ADCC) and decreased binding to complement is expected to decrease the complement-dependent cytotoxicity (CDC) activity of the antibody molecule.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • An antibody molecule of the invention may comprise an Fc with modifications K322A/L234A/L235A or L234F/L235E/P331S (“TM”), which almost completely abolish Fc ⁇ R and C1q binding.
  • An antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) ("LALA mutation").
  • complement activation and ADCC can be decreased by mutation of P329 (position according to EU numbering), e.g., to either P329A or P329G.
  • the antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) and an alanine (LALA-PA) or glycine (LALA-PG) at EU position 329 (position 114 by IMGT numbering). Additionally or alternatively an antibody molecule of the invention may comprise an alanine, glutamine or glycine at EU position 297 (position 84.4 by IMGT numbering).
  • Modification of glycosylation on asparagine 297 of the Fc domain which is known to be required for optimal Fc ⁇ R interaction may confer a loss of binding to Fc ⁇ Rs; a loss of binding to Fc ⁇ Rs has been observed in N297 point mutations.
  • An antibody molecule of the invention may comprise an Fc with an N297A, N297G or N297Q mutation.
  • An antibody molecule of the invention with an aglycosyl Fc domain may be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor, or following the expression of Fc domains in bacteria.
  • IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, giving the IgG a typical half-life of approximately 21 days.
  • Half-life can be extended by engineering the pH-dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4.
  • the T250Q/M428L variant conferred an approximately 2-fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y/S254T/T256E variant (“YTE”), gave an approximately 4-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending half-life may allow the possibility of decreasing administration frequency, while maintaining or improving efficacy.
  • Immunoglobulins are known to have a modular architecture comprising discrete domains, which can be combined in a multitude of different ways to create multispecific, e.g., bispecific, trispecific, or tetraspecific antibody formats. Exemplary multispecific antibody formats are described in Spiess et al., 2015; Kontermann 2012, for example. The antibodies of the invention may be employed in such multispecific formats.
  • the invention provides a humanised or human antibody or antigen-binding fragment thereof, capable of competing with an antibody of the invention described herein (e.g., comprising a set of HCDR and LCDRs of 5E3 (SEQ ID NOS: 3, 4, 5, 12, 13, 14) when defined by Kabat nomenclature) and/or a humanised variant of the V H and V L amino acid sequences of Clone 5E3 (SEQ ID NOs: 2 and 11), for binding to an isolated recombinant human SEMA4A (SEQ ID NO: 613) peptide comprising an epitope, when assessed in a competition assay.
  • an antibody of the invention described herein e.g., comprising a set of HCDR and LCDRs of 5E3 (SEQ ID NOS: 3, 4, 5, 12, 13, 14) when defined by Kabat nomenclature) and/or a humanised variant of the V H and V L amino acid sequences of Clone 5E3 (SEQ ID NOs: 2 and 11), for binding to an
  • the invention provides a humanised or human antibody or antigen-binding fragment thereof, capable of competing for binding to an isolated recombinant human SEMA4A (SEQ ID NO: 613) peptide comprising an epitope, with a clone selected from: a. 5E3 (SEQ ID NOS: 2 and 11) b. 5Hg1-5Lg1 (SEQ ID NOS.: 20 and 29); c. 5Hg1-5Lg2 (SEQ ID NOS.: 38 and 47); d. 5Hg2-5Lg1 (SEQ ID NOS.: 56 and 65); e. 5Hg2-5Lg2 (SEQ ID NOS.: 74 and 83); f.
  • SEMA4A SEQ ID NO: 613
  • 5Hg2-5Lg3 (SEQ ID NOS.: 92 and 101); g. 5Hg2-5Lg5 (SEQ ID NOS.: 110 and 119); h. 5Hg2-5Lg6 (SEQ ID NOS.: 128 and 137); i. 5Hg4-5Lg1 (SEQ ID NOS.: 146 and 155); j. 5Hg4-5Lg2 (SEQ ID NOS.: 164 and 173); k. 5Hg4-5Lg4 (SEQ ID NOS.: 182 and 191); l. 5Hg4-5Lg5 (SEQ ID NOS.: 200 and 209); m.
  • C0120917 (SEQ ID NOS.: 362 and 371); v. C0120918 (SEQ ID NOS.: 380 and 389); w. C0120919 (SEQ ID NOS.: 398 and 407); x. C0120920 (SEQ ID NOS.: 416 and 425); y. C0120921 (SEQ ID NOS.: 434 and 443); z. C0120922 (SEQ ID NOS.: 452 and 461); aa. C0120923 (SEQ ID NOS.: 470 and 479); bb. C0120924 (SEQ ID NOS.: 488 and 497); cc.
  • C0120925 (SEQ ID NOS.: 506 and 515); dd. C0120926 (SEQ ID NOS.: 524 and 533); ee. C0120927 (SEQ ID NOS.: 542 and 551); ff. C0120928 (SEQ ID NOS.: 560 and 569); gg. C0120929 (SEQ ID NOS.: 578 and 587); and hh. C0120930 (SEQ ID NOS.: 596 and 605); wherein the sequences are defined according to Kabat nomenclature and competition for binding is assessed in a competition assay.
  • Competition assays may be selected from cell-based and cell-free binding assays including an immunoassay such as ELISA, homogeneous time resolved fluorescence (HTRF), flow cytometry, fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, bio-layer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays.
  • an immunoassay such as ELISA, homogeneous time resolved fluorescence (HTRF), flow cytometry, fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, bio-layer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays.
  • an immunoassay such as ELISA, homogeneous time resolved fluorescence (HTRF), flow cytometry, fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high
  • An antibody that binds to the same epitope as, or an epitope overlapping with, a reference antibody refers to an antibody that blocks binding of the reference antibody to its binding partner (e.g., an antigen or “target”) in a competition assay by 50% or more, and/or conversely, the reference antibody blocks binding of the antibody to its binding partner in a competition assay by 50% or more.
  • Such antibodies are said to compete for binding to an epitope of interest.
  • An antibody may compete by binding the same epitope as, or an epitope overlapping with, the epitope of a reference antibody.
  • anti-human-SEMA4A humanised or human antibodies described herein can be used to generate an antibody single-chain variable fragment which can then be used to prepare a chimeric antigen receptor (CAR).
  • the antibody single-chain variable fragment is a chimeric protein made up of the light (V L ) and heavy (V H ) chains of immunoglobulins, connected by a short linker peptide.
  • the linker between the VL and VH regions consists of hydrophilic residues comprising glycine and serine to confer flexibility and glutamate and lysine to confer solubility.
  • the antibody single-chain variable fragment can be covalently linked to an intracellular immune cell signaling domain typically through a transmembrane domain to create a CAR.
  • the immune cell signaling domain can be a T-cell, NK cell, macrophage, and/or a myeloid cell domain.
  • An anti-human-SEMA4A humanised or human antibody single-chain variable fragment may be covalently linked to an intracellular T-cell signaling or activation domain, for example via a transmembrane domain to create a CAR. When CARs are expressed in T-cells this provides T cells with the ability to target SEMA4A, in particular human SEMA4A.
  • the invention thus provides CAR T-cells comprising a CAR comprising an anti-human-SEMA4A antibody single- chain variable fragment of an antibody described herein, that binds specifically to SEMA4A, in particular human SEMA4A, for use to treat haematological cancers, for example in MM, NHL, AML, DLBCL or FL patients; methods of treating a patient with a haematological cancer, e.g., MM, , NHL, AML, DLBCL or FL, may comprise administering such CAR T-cells to a patient in need of therapy.
  • a haematological cancer e.g., MM, , NHL, AML, DLBCL or FL
  • the transmembrane domain of a CAR may comprise a hydrophobic alpha helix that spans the cell membrane that anchors the CAR to the plasma membrane, bridging the extracellular antigen recognition domains (i.e., humanised or human antibody single-chain variable fragment) with the intracellular signaling region.
  • the CAR may further comprise a hinge region between the antigen recognition domains and the transmembrane domain.
  • the hinge may serve to enhance the flexibility of the scFv and reduce spatial constraints between the CAR and its target antigen, SEMA4A.
  • the hinge sequence may be based on membrane-proximal regions from immune molecules such as IgG, CD8, and CD28.
  • a CAR of the present disclosure may comprise a CD3-zeta cytoplasmic domain as a CAR endodomain component. T cells require co-stimulatory molecules in addition to CD3 signaling to persist after activation.
  • the endodomain of a CAR may include one or more chimeric domains from co-stimulatory proteins. Signaling domains from a wide variety of co stimulatory molecules have been successfully tested, and may be selected from CD28, CD27, CD134 (OX40), and CD137.
  • the endodomains of CAR receptors may comprise co-stimulatory domains to augment T cell activity, co-stimulatory domains may be selected from those of CD28 or 4-1BB, CD28-4-1BB or CD28-OX40, and cytokines, such as IL-2, IL-5, and IL-12.
  • the invention also provides a nucleic acid or set of nucleic acids encoding an antibody or antigen-binding fragment of the invention, as well as a vector comprising such a nucleic acid or set of nucleic acids. Where the nucleic acid encodes the V H and V L domain, or heavy and light chain, of an antibody molecule of the invention, the two domains or chains may be encoded on the same or on separate nucleic acid molecules.
  • An isolated nucleic acid molecule may be used to express an antibody molecule of the invention.
  • the nucleic acid will generally be provided in the form of a recombinant vector for expression.
  • Another aspect of the invention thus provides a vector comprising a nucleic acid as described above.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell.
  • Vectors may be plasmids, viral e.g., phage, or phagemid, as appropriate.
  • a nucleic acid molecule or vector as described herein may be introduced into a host cell. Techniques for the introduction of nucleic acid or vectors into host cells are well established in the art and any suitable technique may be employed.
  • a range of host cells suitable for the production of recombinant antibody molecules are known in the art, and include bacterial, yeast, insect or mammalian host cells.
  • a preferred host cell is a mammalian cell, such as a CHO, NS0, or HEK cell, for example a HEK293 cell.
  • a recombinant host cell comprising a nucleic acid or the vector of the invention is also provided. Such a recombinant host cell may be used to produce an antigen-binding protein (e.g., antibody) of the invention.
  • an antigen- binding protein e.g., antibody
  • the method comprising culturing the recombinant host cell under conditions suitable for production of the antigen-binding protein, e.g., antibody.
  • the method may further comprise a step of isolating and/or purifying the antigen-binding protein, e.g., antibody.
  • the invention provides a method of producing an antigen-binding protein, e.g., antibody, of the invention comprising expressing a nucleic acid encoding the antigen-binding protein, e.g., antibody, in a host cell and optionally isolating and/or purifying the antigen-binding protein, e.g., antibody, thus produced.
  • Methods for culturing host cells are well-known in the art.
  • Techniques for the purification of recombinant antigen-binding proteins, e.g., antibodies are well-known in the art and include, for example high-pressure liquid chromatograph (HPLC), fast protein liquid chromatograph (FPLC) or affinity chromatography, e.g., using Protein A or Protein L.
  • HPLC high-pressure liquid chromatograph
  • FPLC fast protein liquid chromatograph
  • affinity chromatography e.g., using Protein A or Protein L.
  • purification may be performed using an affinity tag on an antigen-binding protein, e.g., antibody.
  • the method may also comprise formulating the antigen-binding protein, e.g., antibody, into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described below.
  • Antigen-binding proteins, e.g., antibodies, of the invention are expected to find application in therapeutic applications, in particular therapeutic applications in humans, for example in the treatment of a haematological cancers, such as MM, NHL, AML, DLBCL or FL.
  • composition such as a pharmaceutical composition, comprising an ADC, antigen-binding protein, e.g., antibody, or CAR according to the invention and an excipient, such as a pharmaceutically acceptable diluent.
  • the invention further provides an ADC, antigen-binding protein, e.g., antibody, or CAR of the invention, for use in a method of treatment.
  • a method of treating a patient wherein the method comprises administering to the patient a therapeutically-effective amount of an ADC, antigen-binding protein, e.g., antibody, or CAR according to the invention.
  • an ADC, antigen-binding protein, e.g., antibody, or CAR for use in the manufacture of a medicament.
  • a patient as referred to herein, is preferably a human patient.
  • the invention also provides an ADC, antigen-binding protein, e.g., antibody, or CAR of the invention, for use in a method of treating a haematological cancer, such as MM, NHL, AML, DLBCL or FL, wherein the method comprises administering to the patient a therapeutically- effective amount of an ADC, antigen-binding protein, e.g., antibody, or CAR according to the invention.
  • an ADC, antigen-binding protein, e.g., antibody, or CAR for use in the manufacture of a medicament for the treatment haematological cancer, such as multiple myeloma MM, NHL, AML, DLBCL or FL, in a patient.
  • the invention relates to an ADC, antigen-binding protein, e.g., antibody, or CAR of the invention for use in: a) treating, b) delaying progression of, c) prolonging the survival of, and / or (d) providing relief of symptoms in a patient suffering from a haematological cancer, such as MM, NHL, AML, DLBCL or FL.
  • the ADC, antigen-binding protein, e.g., antibody, or CAR as described herein may thus be for use for therapeutic applications, in particular for the treatment of a haematological cancer, such as MM, NHL, AML, DLBCL or FL.
  • An ADC, antigen-binding protein, e.g., antibody, or CAR as described herein may be used in a method of treatment of the human or animal body.
  • an ADC, antigen-binding protein, e.g., antibody, or CAR described herein for use as a medicament (ii) an ADC, antigen-binding protein, e.g., antibody, or CAR described herein for use in a method of treatment of a disease or disorder, (iii) the use of an ADC, antigen-binding protein, e.g., antibody, or CAR described herein in the manufacture of a medicament for use in the treatment of a disease or disorder; and, (iv) a method of treating a disease or disorder in an individual, wherein the method comprises administering to the individual a therapeutically effective amount of an ADC, antigen-binding protein, e.g., antibody, or CAR as described herein.
  • the individual may be a patient, preferably a human patient.
  • Treatment may be any treatment or therapy in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, ameliorating, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of an individual or patient beyond that expected in the absence of treatment.
  • Treatment as a prophylactic measure i.e., prophylaxis
  • prophylaxis is also included.
  • an individual susceptible to or at risk of the recurrence of a haematological cancer such as MM, NHL, AML, DLBCL or FL may be treated as described herein. Such treatment may prevent or delay the reoccurrence of the disease in the individual.
  • an ADC, antigen-binding protein, e.g., antibody, or CAR may be administered alone
  • ADC, antigen-binding proteins, e.g., antibodies, or CAR will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the ADC, antigen-binding protein, e.g., antibody, or CAR.
  • compositions comprising an ADC, antigen-binding protein, e.g., antibody, or CAR as described herein.
  • a method comprising formulating an ADC, antigen-binding protein, e.g., antibody, or CAR into a pharmaceutical composition is also provided.
  • Pharmaceutical compositions may comprise, in addition to the ADC, antigen-binding protein, e.g., antibody, or CAR a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g., human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection or any other suitable route, as discussed below.
  • the pharmaceutical composition comprising the ADC, antigen-binding protein, e.g., antibody, or CAR may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • an ADC, antigen-binding protein, e.g., antibody, or CAR of the invention may be provided in a lyophilised form for reconstitution prior to administration.
  • lyophilised antigen-binding proteins, e.g., antibodies may be reconstituted in sterile water or saline prior to administration to an individual.
  • Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to an individual.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the composition, the type of ADC, antigen-binding protein, e.g., antibody, or CAR, the method of administration, the scheduling of administration and other factors. Prescription of treatment may depend on the severity of the symptoms and/or progression of a disease being treated.
  • a therapeutically effective amount or suitable dose of an ADC, antigen-binding protein, e.g., antibody, or CAR can be determined by comparing in vitro activity and in vivo activity in an animal model.
  • mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the size and location of the area to be treated, and the precise nature of the ADC, antigen-binding protein, e.g., antibody, or CAR.
  • ADC antigen-binding protein
  • CAR CAR-binding protein
  • Haematological malignancies are a diverse group of haematological cancers that affect the blood, bone marrow and lymphatic systems. The main categories are lymphoma, leukaemia, myeloma, myelodysplastic syndromes and myeloproliferative neoplasms.
  • an ADC, antigen-binding protein, e.g., antibody, or CAR as described herein may be for use in a method of treating a haematological cancer, such as MM, NHL, AML, DLBCL or FL.
  • FIG. 1 Primary screening of 70 un-purified humanised IgGs in a Fab-ZAP cell kill assay in NCI-H929 cells identified 18 clones that showed a cell kill of > 30%. All 70 unpurified clones were screened at a final concentration of 1.56%. 12 out of 18 hit clones (•) were re-expressed and purified for further characterisation. 10 out of the 12 clones selected for progression also showed inhibition in the 5E3 epitope competition assay (see Figure 2).
  • Figure 2 Primary screening of 320 un-purified humanised IgGs in an epitope competition assay in MM.1S cells identified 17 clones that showed an inhibition of > 30%. All 320 un-purified clones were screened at a final concentration of 25%. 10 out of 17 hit clones (•) were re-expressed and purified for further characterisation. Two clones that showed ⁇ 30% inhibition were also re-expressed and purified for further characterisation: 5Hg2/5Lg6 and 5Hg4/5Lg4. All 12 clones selected for further characterisation were also identified as hits in the Fab-ZAP cell kill assay.
  • Figure 3 Primary screening of un-purified fully human phage display IgGs in a Fab-ZAP cell kill assay in NCI-H929 cells identified a panel of clones that showed a cell kill of > 15%. All un-purified clones were screened at a final assay concentration of 3.13%. 10 clones (•) were re-expressed and purified for further characterisation.
  • Figure 4 Primary screening of un-purified fully human phage display IgGs in a binding assay in MM.1S cells identified clones that bound with a fluorescence intensity >1 million FLU. All un-purified clones were tested at a final assay concentration of 1.56%. Binding assay data was used in conjunction with Fab-ZAP assay data ( Figure 3) to select a panel of 10 clones (•) that were hits in the Fab-ZAP assay and showed good binding (> 1 million FLU) to MM.1S cells. C0120910 was progressed as it did show binding when tested at a final assay concentration of 25% (data not shown).
  • Figure 5 Measuring binding specificity of humanised leads (A) and fully human phage display leads (B) expressed in the human IgG 1 Alya vector.
  • the ability of leads to bind to expi293 cells transiently transfected with human SEMA4A (i), mouse SEMA4A (ii), cyno SEMA4A (iii), human SEMA4B (iv) and mock transfected expi293 cells (v) was measured using the Mirrorball. Binding of leads was compared to mouse 5E3 expressed as human lgG1 Alya. C0021144 IgG was included as a positive control for binding to mock transfected expi293 cells.
  • a commercially available anti SEMA4B polyclonal antibody (R&D Systems, AF5485) was included as a positive control for binding to human SEMA4B transfected expi293 cells.
  • human IgG1 Alya vector see example 4.1.
  • Figure 6. Measuring the ability of humanised leads (A) and fully human leads (B) expressed in the human IgG1 Alya vector to kill NCI-H929 cells in a Fab-ZAP cell kill assay. Cell kill ability of all clones was compared to mouse 5E3 in the human IgG1 Alya vector. A number of humanised clones showed IC 50 values comparable to the mouse 5E3 antibody (V H and V L SEQ ID NOS: 2 and 11).
  • humanised clones did not demonstrate complete cell kill.
  • human Alya IgG1 vector see example 4.1.
  • Mouse 5E3 expressed as a human isotype (human IgG1 Alya) was competed into the assay and test clones were compared against this. All humanised clones showed some level of competition with 5E3.
  • Figure 8. Epitope binning.
  • the OctetRED (Pall ForteBio) instrument was used as described in section 4.6 to perform epitope binning, grouping lead anti-human SEMA4A clones into bins based upon binding to recombinant human SEMA4A.
  • the antigen biotinylated recombinant human SEMA4A
  • FIG. 8A is representative of data with fully human clones that have overlapping or competing epitopes with 5E3.
  • Figures 8B-D show data corresponding to fully human clones with non-overlapping or non-competing epitopes with 5E3 (B) and (C) and with each other (D).
  • Figure 9. Measuring binding specificity of humanised leads (A) and fully human leads (B) expressed in the human IgG1 Alya vector and conjugated to McMMAF.
  • Humanised clones showed a range of IC50 values across cell lines, with no cell kill observed in K562 cells, a negative control cell line for SEMA4A expression.
  • human Alya IgG1 vector see example 4.1. J6M0 anti-BCMA monoclonal antibody control (Tai et al., 2014).
  • Figure 11 Measuring the ability of fully human leads conjugated to McMMAF expressed in the human Alya IgG1 vector to kill NCI-H929 (A), MM1.S (B) and K562 (C) cells in a cell kill assay. Cell kill ability of all clones was compared to mouse 5E3 as Alya IgG1 conjugated to McMMAF.
  • the Mean Stain Integrated Intensity was also extracted for each concentration and time point and plotted as a fold change in signal over baseline, as is shown in panel (D).
  • panel (D) For a description of human Alya IgG1 vector see example 4.1.
  • Figure 13 Measuring binding affinities (KD) of SEMA4A specific human antibodies (Alya IgG1 conjugated to McMMMAF) by flow cytometry. KD values for all clones were compared to mouse 5E3 as human Alya IgG1 and McMMAF coupled. Humanised leads, 5Hg1/5Lg1 and 5Hg2/5Lg2 showed similar KD values to mouse 5E3. The fully human, phage display derived leads had lower affinities than mouse 5E3.
  • the lower limit of detection was determined as the average of the blank sample plus three standard deviations of the blank. The limit of detection was calculated as the lowest concentration tested (1.56 ng/mL) above the LLOD.
  • B sSEMA4A in healthy and myeloma patient serum was detected by ELISA and found to be 3.3 ng/ml and 8.1 ng/ml respectively.
  • Figure 16 Assessing the impact of soluble SEMA4A on the potency of mouse 5E3 and fully human leads in a cell kill assay using NCI-H929 cells.
  • a concentration of up to 100 ng/ml sSEMA4A had no significant impact on the ability of either mouse 5E3 or the fully human clones, each as Alya IgG1 conjugated with McMMAF, to kill NCI-H929 cells in terms of IC50 values.
  • human Alya IgG1 vector see example 4.1.
  • VH and VL gene sequence was synthesised de novo and cloned into the appropriate IgG expression vector (Persic, et al., 1997) using standard molecular biology techniques.
  • 5E3 Grafts Heavy Chain
  • 5E3 Grafts (Lambda Light Chain) ID Closest Human Germline Notes Table 4.
  • Human V H and V L germline sequences chosen as candidate frameworks for 5E3 humanisation.
  • 10x V H and 7x V L human germlines were chosen based on their overall similarity to the original mouse 5E3 sequence. All genes were synthesised de novo (Genewiz).
  • the combined DNA was diluted in OptiMEM and used to transfect Expi293TMF cells in a 96 deepwell block (Merck, cat: AXYPDW20CS) using ExpiFectamine as transfection reagent.
  • Cells were grown 37°C/8% CO 2 /80% humidity shaking at 1000rpm and fed (using enhance 1 and enhance 2) 18 hours post transfection.
  • IgGs were harvested 4 days post transfection and used as crude, un-purified IgGs in subsequent assays.
  • Example 2 Isolation of fully human anti-SEMA4A antibodies from na ⁇ ve libraries by phage display 2.1 Production of recombinant human, cynomolgus and mouse SEMA4A Recombinant human SEMA4A (UniProt ID Q9H3S1, amino acids 33 - 683), recombinant cynomolgus SEMA4A (UniProt ID G7NV79, amino acids 32 – 679) and recombinant mouse SEMA4A (UniProt ID Q62178, amino acids 33-682) with C-terminal Avi tag and His10 tag (SEQ ID NOS: 616, 618, 617, respectively) were cloned into pcDNA3.1 vector and transiently expressed in the Expi293 expression system.
  • SEMA4A UniProt ID Q9H3S1, amino acids 33 - 683
  • recombinant cynomolgus SEMA4A UniProt ID G7NV
  • the proteins were subsequently purified using HisTrap HP His tag protein purification columns (40 mM Imidazole wash, 400 mM Imidazole elution).
  • the proteins (human SEMA4A at 75 kDa, cynomolgus SEMA4A at 79kDa and mouse SEMA4A at 79kDa) were buffer exchanged into 50 mM Bicine pH 8.3 and biotinylated using BirA biotin ligase (3 mg of biotin ligase per 10 nmol of substrate protein).
  • the proteins were finally buffer exchanged into Dulbecco’s phosphate buffered saline (DPBS) with 5% (w/v) Trehalose (pH 7).
  • DPBS Dulbecco’s phosphate buffered saline
  • Anti-SEMA4A scFv antibodies were isolated from the phage display libraries using a series of selection cycles on recombinant, biotinylated human and mouse SEMA4A protein (avi-SEMA4A-His10, made in house) essentially as previously described (Hawkins et al., 1992; Vaughan et al., 1996).
  • biotinylated human SEMA4A in DPBS pH 7 was added (final concentration of 100 nM biotinylated human SEMA4A) to purified phage particles that had been pre- incubated for 1 hour in Marvel-PBS (3% w/v) containing Streptavidin-coupled paramagnetic beads (Dynabeads ® M280, Invitrogen Life Sciences, UK). Streptavidin beads were removed prior to addition of antigen.
  • Phage particles that bound to the biotinylated human SEMA4A were captured using new Streptavidin-coupled paramagnetic beads, and weakly-bound phage were removed by a series of wash cycles using PBS-Tween (0.1% v/v). Bound phage particles were eluted from the beads using Trypsin (10 ⁇ g/ml final concentration diluted in 0.1 M sodium phosphate buffer; pH 7), infected into E. coli TG1 bacteria and rescued for the next round of selection (Vaughan et al., 1996).
  • biotinylated SEMA4A antigen specifically 50 nM of biotinylated human or mouse SEMA4A at round 2 and 25 nM of biotinylated human SEMA4A at round 3.
  • Mouse antigen was introduced at round 2 to drive for mouse cross-reactivity.
  • Binding of un- purified scFv was detected using two antibodies – a mouse anti c-myc antibody (Bio-Rad, US; cat: MCA2200GA) binding to a myc tag expressed on the scFv followed by an anti-mouse antibody conjugated to Alexa Fluor 647 (Invitrogen, US; cat: A21235). Selection outputs were screened at a single concentration as un-purified bacterial periplasmic extracts containing scFv, prepared in 200 mM tris buffer pH 7.4, 0.5 mM EDTA and 0.5 M sucrose.
  • Clones binding non-specifically were identified using a concurrent assay with expi293 cells that had been mock transfected (i.e., human SEMA4A DNA was omitted during transfections). All dilutions were performed in Hanks’ balanced salt solution (Sigma, UK; cat: H8264) containing 0.1% bovine serum albumin (BSA) (Sigma, UK; cat: A9576) (assay buffer). Assay plates were incubated at room temperature in the dark for 4 hours prior to reading on a Mirrorball fluorescence cytometer (SPT Labtech, UK) using a 640 nm laser for excitation and measuring emission in the FL-4 channel (650 and 690 nm). Data was analysed as Count x median mean intensity (x FLU).
  • the scFvs were batch converted into IgGs, firstly replacing the (G 4 S) 3 scFv linker with a DNA segment coding for the hinge and constant domains for Heavy Chain (HC) and the promoter and signal sequence for Light Chain (LC) and secondly replacing the scFv backbone with the LC constant domains and the HC signal sequence in the IgG vector (as described in Xiao et al., 2017).
  • the final expression vector is bicistronic with both HC and LC under the control of a CMV promoter.
  • the converted IgGs were subsequently expressed as crude IgGs as described in section 3.2.
  • Example 3 Functional screening of un-purified 5E3 humanised and fully human, phage display derived, IgG1s – in primary screens
  • Example 3 describes the primary screening of un-purified IgG1s (section 1.2) from the 5E3 humanisation approach (Example 1) and from the phage display approach (Example 2). Both sets of IgGs were screened for function in a Fab-ZAP cell kill assay.
  • the 5E3 humanisation IgGs were also screened for their ability to compete for binding to the 5E3 epitope in a mouse 5E3 epitope competition assay.
  • the fully human, phage display derived, clones were also screened for their ability to bind to MM.1S multiple myeloma cells that endogenously express human SEMA4A.
  • the Fab-ZAP assay assessed the ability of the un-purified IgGs to bind, internalize, and kill NCI-H929 multiple myeloma cells in vitro using a secondary-saporin conjugate, Fab-ZAP (Advanced Targeting Systems, San Diego, CA, IT-51), CellTiter Glo 2.0 (Promega, Madison, WI, G9248) and a Pherastar plate reader (BMG Labtech, Germany).
  • Fab-ZAP is an anti- human Fab that has been conjugated to the warhead saporin.
  • Un-purified humanised 5E3 and fully human, phage display derived, human IgG1s were pre-diluted 8-fold and 4-fold, respectively.
  • the un-purified human IgG1s were contained in Expi293 expression medium (Gibco, UK, A1435101). Following an 8-fold dilution into the assay, final assay concentrations were 1.56 and 3.13%, respectively.
  • the 5E3 epitope competition assay assessed the ability of the un-purified IgGs expressed in a human IgG1 backbone to compete for binding to the mouse 5E3 epitope on human SEMA4A endogenously expressed by MM.1S cells, a human multiple myeloma cell line.
  • the assay utilised a secondary-Alexa Fluor 647 conjugate, 5E3 expressed in a mouse IgG1 ( ⁇ ) backbone (BioLegend ® , San Diego, CA , 148402) and a Mirrorball fluorescence cytometer (SPT Labtech, UK).
  • Binding of 5E3 mouse IgG1 to MM.1S cells was measured via an anti-mouse secondary labelled with Alexa Fluor 647 (Invitrogen, A21235) in the presence of a single concentration of each un-purified human IgG1. Occupation of the 5E3 epitope on SEMA4A by the un-purified IgG resulted in a reduction in the fluorescence signal, as measured by the Mirrorball.
  • the MM.1S binding assay assessed the ability of the un-purified human IgG1s to bind directly to human SEMA4A that was endogenously expressed by MM.1S cells. Direct binding of a single concentration of un-purified human IgG1s to MM.1S cells was measured via an anti- human secondary labelled with Alexa Fluor 647 (Invitrogen, A21445).
  • Example 4 Secondary screening of purified human Alya IgG1 4.1 Reformatting of SEMA4A binding antibodies to human Alya IgG1 Specific variable fragments were expressed as a human IgG1 – ADC isotype ( Alya IgG1) which consists of human IgG1 constant chain with modifications to introduce 2 cysteines into each heavy chain fragment to which drug molecules can be covalently conjugated – this results in a ‘drug-to-antibody’ ratio of four (4DAR). Variable heavy chain (V H ) and variable light chain (V L ) domains into vectors expressing whole human antibody heavy and light chains respectively.
  • V H Variable heavy chain
  • V L variable light chain domains into vectors expressing whole human antibody heavy and light chains respectively.
  • variable heavy chains were cloned into a mammalian expression vector (pEU1.22, Alya vector) containing the human heavy chain constant domains and regulatory elements to express whole IgG1 heavy chain in mammalian cells.
  • pEU1.22 is engineered to contain two site-specific cysteines that enable controlled drug conjugation (239iCys and S442C (EU numbering) in the CH2 and CH3 Fc domain, respectively).
  • the variable light chain domain was cloned into a mammalian expression vector for the expression of the human lambda light chain constant domains (pEU4.4) or human kappa light chain constant domains (pEU3.4) and regulatory elements to express whole IgG light chain in mammalian cells.
  • the vector for expression of heavy chain was originally described by Thompson et al., 2016. Vectors for the expression of light chains were originally described in Persic, et al., 1997.
  • the heavy and light chain IgG expression vectors were transiently transfected into ExpiCHO (ThermoScientific UK; cat. number: A29133) cells where the antibody was expressed and secreted into the medium.
  • Harvested media was filtered prior to purification.
  • the IgGs were purified using Protein A chromatography (HiTrap Fibro PrismA, Cytiva, UK). Culture supernatants were loaded onto an appropriate Protein A column pre- equilibrated in 25 mM Tris pH 7.4, 50 mM NaCl.
  • Bound IgG was eluted from the column using 0.1 M Sodium Citrate pH 3.0, 100 mM NaCl.
  • the IgGs were buffer exchanged into PBS.
  • the purified IgGs were passed through a 0.2 ⁇ m filter and the concentration of IgG was determined by absorbance at 280 nm using an extinction coefficient based on the amino acid sequence of the IgG.
  • the purified IgGs were analysed for aggregation or degradation using SEC-HPLC and SDS-PAGE techniques.
  • a further modification to the assay was the substitution of mouse anti c-myc antibody and anti- mouse antibody conjugated to Alexa Fluor 647 with anti-human Alexa Fluor 647 detection reagent (ThermoFisher Scientific, UK; cat: A21445) as the recombinant human Alya IgG1 had no c-myc tag.
  • the required binding profile was for binding to human SEMA4A, mouse SEMA4A, cyno SEMA4A but not to human SEMA4B and mock transfected expi293 cells. The results of this experiment are shown in Figure 5.
  • Table 6 Cell kill ability of humanised lead clones expressed in the human Alya IgG1 vector compared to mouse 5E3 IgG1 Alya in a FabZAP assay.
  • IgG competition assay to assess epitope binding of SEMA4A specific Alya IgGs (purified, unconjugated) Hits from the primary screening were re-formatted into the human Alya IgG1 vector and re- expressed and purified as described in section 4.1.5E3 epitope competition assay methods are described in section 3.2 and were used to test purified human Alya IgG1s.
  • the purified human Alya IgG1s were diluted to a 4x stock and then serially diluted using 4-fold dilutions to generate a 10 point concentration response curve.10 ⁇ l of the concentration response curve were transferred to the assay plate in duplicate in place of un-purified IgG. The results of this experiment are shown in Figure 7 and Table 7.
  • scFvs were also converted to Fab fragments, sub-cloning the Variable heavy chain (V H ) and variable light chain (V L ) domains into vectors expressing part of human antibody heavy and the whole light chains respectively.
  • the variable heavy chains were cloned into a mammalian expression vector (pEU1.3 Fab) that contains the CH1 human heavy chain constant domain, part of the hinge region, and required regulatory elements to express the Fab heavy chain fragment in mammalian cells.
  • variable light chain domain was cloned into a mammalian expression vector for the expression of the human lambda light chain constant domains (pEU4.4) or human kappa light chain constant domains (pEU3.4) and regulatory elements to express whole IgG light chain in mammalian cells.
  • pEU4.4 human lambda light chain constant domains
  • pEU3.4 human kappa light chain constant domains
  • the Fab heavy and light chain IgG expression vectors were transiently transfected into ExpiCHO cells (ThermoScientific UK; cat. number: A29133) where the Fab fragments was expressed and secreted into the medium. Harvested media was filtered prior to purification.
  • the Fab fragments were purified using an affinity matrix recognising the CH1 domain of human IgG antibodies (Capture Select CH1-XL Columns, ThermoFisher Scientific, UK). Culture supernatants were loaded onto an appropriate Capture Select column pre-equilibrated in PBS. Bound Fab fragment was eluted from the column using 50mM Sodium Acetate buffer pH 4.0 – 4.5. The Fab fragments were buffer exchanged into PBS. The purified Fab fragments were passed through a 0.2 ⁇ m filter and the concentration of the Fab fragments was determined by measuring their absorbance at 280 nm using an extinction coefficient based on the amino acid sequence of the Fab fragment.
  • the purified Fab fragments were analysed for aggregation or degradation using SEC-HPLC and SDS-PAGE techniques.
  • the OctetRED (Pall ForteBio) instrument was used to assess the kinetic parameters of the interactions between the lead anti-SEMA4A IgGs and recombinantly produced human SEMA4A and cynomolgus SEMA4A.
  • the Octet biosensor uses an optical analytical technique that analyses the interference pattern of white light reflected from two surfaces: a layer of immobilised protein on the sensor tip, and an internal reference layer.
  • any changes in binding at the biosensor tip result in a shift in interference pattern, which can be measured in real time.
  • Molecules associating with or dissociating from ligands at the biosensor tip shift the interference pattern and generate a response on the Octet system which is recorded by the acquisition software.
  • a defined concentration of the analyte species is brought into contact with the coupled ligand and any binding is detected as an increase in signal (association phase). This is followed by a period of buffer rinse, during which dissociation of the analyte species from the surface immobilised ligand can be observed as a decrease in signal (dissociation phase). Repetition of this with a range of analyte concentrations provides data for the analysis of binding kinetics.
  • An Octet Kinetics buffer (PBS containing 0.01% (v/v) BSA and 0.002% (v/v) Tween20) is typically used as the diluent buffer for the analyte samples and as the flow buffer during the dissociation phase.
  • the experimental data is recorded as shift in interference pattern (nm) over time, which is directly proportional to the optical thickness at the biosensor tip, which in turn is an approximate measure of the mass of analyte bound.
  • the proprietary Octet Data Analysis software package can then be used to process data and fit binding models to the data sets. Returned association (ka, M-1 s-1) and dissociation (kd, s-1) rate constants allow calculation of dissociation (K D , M) affinity constants.
  • the affinity of binding between the analytes (humanised and phage display derived IgGs as human Alya IgG1s and Fabs) and human SEMA4A and cynomolgus SEMA4A, was estimated using assays in which the biotinylated SEMA4A antigen was captured on a streptavidin sensor tip. A fresh sensor tip was used for each measurement and no regeneration was used.
  • the antigen (biotinylated recombinant human SEMA4A) immobilized onto a streptavidin biosensor is presented to the two competing analytes in consecutive steps. Binding to distinct non-overlapping epitopes is indicated if saturation with the first Fab fragment (mouse 5E3) does not block binding of the second IgG. A defined concentration of the analyte species is brought into contact with the coupled ligand and any binding is detected as an increase in signal (association phase).
  • Epitope binning is achieved by saturating this first association (determined during affinity measurements, typically 5 minutes), followed by a second association step where the sensor is dipped into a well containing both the first analyte (mouse 5E3 Fab) as well as a second analyte (na ⁇ ve SEMA4A IgG) (typically 5 minutes).
  • the first analyte is included to maintain saturation whilst the second analyte attempts to bind. This is followed by an appropriate length of dissociation time (typically 10 minutes).
  • a fresh sensor tip was used for each measurement and no regeneration was used.
  • Figure 8A is representative of data generated from two clones that have overlapping or competing epitopes.
  • Figures 8B-D show data corresponding to clones with non-overlapping epitopes.
  • Example 5 Conjugation of linker and warhead (McMMAF) to human Alya IgG1 5.1 Conjugation of SEMA4A specific human Alya IgG1s to McMMAF Humanised and fully human leads were cloned into the human Alya IgG1 vector and expressed, purified and concentrated to 10mg/ml in DPBS, pH 7. Each protein was mildly reduced using TCEP (Tris-(2-carboxyethyl)-phosphine Hydrochoride, Sigma cat: 75259) at a 40-fold molar excess of TCEP to antibody for 3 hours at 37 ° C.
  • TCEP Tris-(2-carboxyethyl)-phosphine Hydrochoride
  • each conjugated SEMA4A IgG human Alya IgG1 was buffer exchanged into PBS, pH 7.2, 1 mM EDTA, concentrated and purified using Superose 6 Increase 10/300 GL (Sigma-Aldrich, cat: GE29-0915-96) using PBS, pH 7.2, 1 mM EDTA.
  • the IgGs were run on SDS PAGE (NuPAGE 4-12% BisTris gels (12 well), ThermoFisher Scientific, cat: NP0322PK2) at 200V for 60 minutes alongside each unconjugated SEMA4A human Alya IgG1 to confirm conjugation.
  • the human Alya IgG1 clones were conjugated to McMMAF as described in section 5.1.
  • the capacity of the conjugates to kill all four cell lines was tested and benchmarked relative to an McMMAF-conjugated BCMA- specific Alya IgG1, known as J6M0 (Tai et al., 2014).
  • the three cell lines were selected for these assays as high (MM.1S), medium (NCI-H929) and low (Karpas-25) expressors of human SEMA4A.
  • K562 cells do not express human SEMA4A.
  • NCI-H929 cells were shown to express equivalent levels of both targets (3E5 antibody binding sites per cell for both SEMA4A and BCMA, determined using QuantumTM Simply Cellular® anti-Mouse IgG beads cat# 815, Bangs Laboratories, Inc, Indiana, USA).
  • This cell line was considered to be a suitable line to understand the potency of the SEMA4A panel of mAbs versus J6M0 for cell kill and internalisation rates.
  • the McMMAF conjugates were diluted to a 4x stock and then serially diluted using 4-fold dilutions to generate a 12-point concentration response curve.
  • This assay was set up using known concentrations of McMMAF-conjugated Alya IgGs, Fab- pHast (Advanced Targeting Systems, San Diego, CA, PH-01), Hoechst 33342 (Invitrogen, UK, H3570) and an ImageXpress Micro high content imaging system (Molecular Devices, San Jose, CA).
  • Fab-pHast is an anti-human secondary conjugated to a pH-sensitive fluorescent label. The neutral pH of tissue culture media quenches Fab-pHast fluorescence. It is only when the antibody-secondary complex is internalised to the acidic compartments of the endocytic pathway that significant Fab-pHast fluorescence can be detected.
  • the humanised and fully human lead panel as McMMAF conjugated human Alya IgG1, were labelled by dilution to a known concentration in 10x Fab-pHast solution. A serial dilution was set up in the same solution to create a titration curve, before incubating the labelling reactions at room temperature for 20 minutes. Labelled antibodies were added at a 1 in 10 ratio to NCI- H929 cells that had previously been seeded in serum free RPMI 1640 (Gibco, UK, A10491- 01) on Poly-D-Lysine-coated 96-well tissue culture plates.
  • the treated cells were incubated at 37°C/5% CO 2 /95% humidity and imaged on the blue and yellow channels of the ImageXpress Micro at 3, 6 and 24 hours. These images were processed using the MetaXpress 6 software package (Molecular Devices, San Jose, CA) to identify objects in the blue (Hoescht staining of all nuclei) and yellow (Fab-pHast label that has been internalised) channels based on defined thresholds.
  • the software reported both the percentage of Fab-pHast positive cells and their Mean Integrated Intensity (defined as the sum of all Fab-pHast positive pixel intensities divided by the total number of Hoescht positive nuclei).
  • a range of antibody concentrations were evaluated using a 10-point, 3-fold dilution series.
  • Cells were then incubated with Alexa Fluor 647 conjugated AffiniPure Fab Fragment Goat Anti-Human IgG (H+L) (Jackson Immunoresearch Europe Limited, Ely, Cambridgeshire) diluted 1:100, at 4°C for 1 hour, followed by two washes in FACS buffer.
  • Cells were then stained with LIVE/DEADTM Fixable Violet Dead Cell Stain (Life Technologies, L34955) for 30 minutes at 4°C, followed by two washes in FACS buffer. Cells were then fixed using BD Cell Fix (BD Biosciences, 340181).
  • Example 6 Germlining fully human, phage display derived leads and verification of retained function 6.1 Germlining fully human, phage display derived leads Fully human leads identified from phage display selections were germlined to help mitigate potential immunogenicity risks. Individual antibody sequences were compared to human germline V, D and J regions accessible via IMGT (ImMunoGeneTics; www.imgt.org) or ImmuneDiscover databases. Amino acid residues differing from the canonical human germline sequence that resided outside of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3 regions and which were not Vernier residues, were reverted.
  • White maxisorp plates (ThermoScientific, UK, 436110) were coated with 50 ⁇ l of capture antibody (mouse anti-human SEMA4A clone 5E3, BioLegend ® , San Diego, CA, 148402) at 5 ⁇ g/mL in PBS and incubated overnight at 4°C. Unbound antibody was removed and plates washed with PBS. Non-specific binding was blocked by incubation with casein (ThermoScientific, UK, 37528) for one hour at room temperature, followed by a PBS wash. 50 ⁇ l per well of the unknown sample or the diluted standard (recombinant human SEMA4A (Abcam, UK, ab182683)) were incubated for one to two hours at room temperature.
  • capture antibody mouse anti-human SEMA4A clone 5E3, BioLegend ® , San Diego, CA, 148402
  • Unbound antibody was removed and plates washed with PBS. Non-specific binding was blocked by incubation with case
  • sSEMA4A was derived from the cell culture media (CCM) of NCI-H929 cells that had been concentrated 100-fold. sSEMA4A levels in the CCM were quantified using the ELISA described in Example 7.1.
  • CCM containing no sSEMA4A was derived from NCI-H929 SEMA4A knockout cells and was used for the 0 ng/ml SEMA4A concentration.
  • Cell kill IC50 values of the fully human, phage display derived, human Alya IgG1s conjugated to McMMAF showed no significant changes compared to IC50 values measured in the absence of sSEMA4A.
  • SEMA4A Sections of formalin-fixed, paraffin-embedded (FFPE) tissues were cut at 4 microns thickness. Using a Ventana Discovery autostainer, sections were deparaffinised, and antigen retrieval was performed in cell conditioning solution or CC1 (low pH) solution at 95°C for 64 minutes. An antibody against SEMA4A (Atlas antibodies, HPA069136, rabbit polyclonal) was applied at 0.2 ⁇ g/ml concentration for 60 minutes.
  • FFPE 3,3′-Diaminobenzidine
  • hematoxylin hematoxylin
  • a range of FFPE cell pellets comprising cell lines with known SEMA4A expression, including cells overexpressing its closest family member SEMA4B, were used to optimise the staining protocol and confirm its specificity.
  • Normal human tissue with expected SEMA4A expression (tonsil, spleen) was used to confirm the suitability of the assay for FFPE tissue.
  • BCMA Sections of formalin-fixed, paraffin-embedded (FFPE) tissues were cut at 4 microns thickness.
  • positive staining for human SEMA4A was observed in immune cells (consistent with B-cells as well as certain cells of myeloid/dendritic lineage), as well as in some cells of the brain.
  • tumour tissue a high prevalence of positive staining was observed in samples of MM (92% of samples with some positive tumour staining), DLBCL (100%), FL (100%) and AML (74%). Expression intensity was strongest overall in MM and DLBCL.
  • SEMA4A is a potentially valuable target for a specific antibody-based anti-tumour therapeutic approach.
  • SEMA4A expression in multiple myeloma and DLBCL was compared with that of other potential therapeutic targets for these tumours, namely BCMA for MM and CD22 for DLBCL.
  • BCMA for MM
  • CD22 for DLBCL.
  • the same samples stained for SEMA4A were stained and scored for BCMA or CD22 expression, using the same scoring system. High expression was defined as a score ⁇ 6. Results for MM are shown in Tables 13 and 14, and those for DLBCL in Tables 15 and 16. All MM samples had some positivity for both SEMA4A and BCMA.
  • the JJN-3 xenograft model of human MM is established in female athymic nude mice (Envigo, Frederick, MD) by implanting 10 ⁇ 10 6 cells (for example) subcutaneously in the flank. Tumour growth can be monitored over the course of the study, and tumour volume calculated as [length (mm) ⁇ width (mm) x width (mm)]/2.
  • the treatment information is not blinded during tumour measurement.
  • Mouse body weight and tumour measurements are determined twice weekly for the duration of the study. Sample size estimates for 100 percent regression in tumour volume (compared to control) are calculated using nQuery version 4.0 (for example) (Statistical Solutions Ltd., Cork, Ireland, 2015).
  • Kupchan SM Structural requirements for antileukemic activity among the naturally occurring and semisynthetic maytansinoids. J Med Chem. 1978 Jan;21(1):31-7. doi: 10.1021/jm00199a006. PMID: 563462. 16.

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Abstract

L'invention concerne un conjugué anticorps-médicament (ADC) comprenant un anticorps humanisé ou humain anti-SEMA4A humain, un lieur et une cytotoxine.
EP22843860.2A 2021-12-11 2022-12-12 Immunothérapie pour le cancer Pending EP4444751A1 (fr)

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GB8308235D0 (en) 1983-03-25 1983-05-05 Celltech Ltd Polypeptides
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
JPS61134325A (ja) 1984-12-04 1986-06-21 Teijin Ltd ハイブリツド抗体遺伝子の発現方法
GB8607679D0 (en) 1986-03-27 1986-04-30 Winter G P Recombinant dna product
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
US6441163B1 (en) 2001-05-31 2002-08-27 Immunogen, Inc. Methods for preparation of cytotoxic conjugates of maytansinoids and cell binding agents
EP1521769B1 (fr) 2002-07-09 2015-09-09 Dömling, Alexander Conjugues de tubulysine
AU2003266233A1 (en) 2002-07-09 2004-01-23 Morphochem Aktiengesellschaft Fur Kombinatorische Chemie Novel tubulysin analogues
DE10254439A1 (de) 2002-11-21 2004-06-03 GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) Tubulysine, Herstellungsverfahren und Tubulysin-Mittel
WO2008103474A1 (fr) 2007-02-20 2008-08-28 Anaptysbio, Inc. Procédés de production de pharmacothèques, et leurs utilisations
WO2009134279A1 (fr) 2007-07-20 2009-11-05 The Regents Of The University Of California Analogues de la tubulysine d
EP2181101A2 (fr) 2007-07-20 2010-05-05 Helmholtz-Zentrum für Infektionsforschung GmbH Analogues de la tubulysine d
CN101909441B (zh) 2007-10-25 2015-05-13 恩多塞特公司 微管蛋白抑制剂及其制备方法
JP2013535220A (ja) 2010-08-06 2013-09-12 エンドサイト，インコーポレイテッド ツブリシンを調製するためのプロセス
MX2014004226A (es) * 2011-10-06 2014-08-27 Univ Texas Anticuerpos anti-sema4a de humano útiles para tratar enfermedades.
UY36075A (es) 2014-04-11 2015-10-30 Medimmune Llc Derivados de tubulisina
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